Phoenixin Peptides

ABSTRACT

Human phoenixin peptides, analogs and mimetics useful in production of anti-phoenixin antibodies, diagnostic screening and assays, and in modulating cellular concentration of cAMP, and treatment of disorders related to cAMP or Ca 2+  concentration in cells, modulating hypertension and cardiovascular function, modulating gonadotrophs and gastric emptying.

This application is the United States National Stage of InternationalPatent Cooperation Treaty Patent Application No. PCT/US2012/039743,filed May 25, 2012, which claims the benefit of U.S. Provisional PatentApplication No. 61/519,746, filed May 28, 2011, and U.S. ProvisionalPatent Application No. 61/519,747, filed May 26, 2011, each herebyincorporated by reference herein.

I. TECHNICAL FIELD

Human phoenixin peptides, analogs and mimetics useful in production ofanti-phoenixin antibodies, diagnostic screening and assays, and inmodulating cellular concentration of cAMP, and methods of regulating, ortreatment of disorders benefited by peptides capable of regulating, cAMPconcentration in cells, modulating hypertension cardiovascular function,modulating gonadotrophs and gastric emptying.

II. BACKGROUND

The human propeptide Swiss-Prot: Q8N5G0 (also referred to as “Q8N5G0”)including 168 amino acids as shown in FIG. 1 (SEQ. ID NO.: 1) may beprocessed to obtain peptide forms which have been shown to modulate cellhomeostasis of cardiovascular response, blood pressure, gastricemptying, and smooth muscle response.

However, all the processed forms of the human propeptide Q8N5G0 have notyet been fully identified or described and active chemically synthesizedpeptides having a reduced number of resides, greater stability or havinggreater activity over those known prior to the instant invention wouldbe useful for: the production of polyclonal and monoclonal antibodies;diagnostic screening and assays; modulation of biochemical pathways;regulating concentration of cAMP or Ca²⁺ in cells, smooth muscleresponse, gastric emptying; or treatment of disorders treatable withpeptides which can modulate concentration of cAMP or Ca²⁺ in cells,smooth muscle response, gastric emptying, or the like.

II. SUMMARY OF THE INVENTION

Accordingly a broad object of the invention can be to provide novelpurified and isolated native peptides or chemically synthesized purifiedand isolated peptides (also referred to as the “phoenixin peptides”)each corresponding to a portion, or providing in whole or in part amimetic, of the human propeptide Q8N5G0) comprising 168 amino acids asshown in FIG. 1 (SEQ. ID NO.: 1)(or similar propeptide of other speciesas shown in FIG. 71 (SEQ. ID NOS.: 61 through 66)(individually andcollectively referred to herein as the “phoenixin propeptide”). Nativephoenixin peptides can be identical between species such as human,mouse, rat, pig, bovine, and canis. The purified and isolated nativephoenixin propeptide and chemically synthesized isolated phoenixinpeptides can be useful in regulating one or more of: production of cAMPin cells, homeostasis of cardiovascular responses, blood pressure,gastric emptying, and smooth muscle response, or useful for treatment ofdisorders that are benefited by regulation one or more of: production ofcAMP in cells, homeostasis of cardiovascular responses, blood pressure,gastric emptying, and smooth muscle response.

Another broad object of the invention can be to provide chemicallysynthesized purified and isolated phoenixin peptides soluble andsufficiently stable in aqueous solutions, tissues, tissue homogenates,cell cultures, eluted fractions containing components thereof, or thelike, useful screening assays and diagnostic procedures related todetermination of one or more of: levels of phoenixin propeptides, levelsof native phoenixin peptides resulting from processing of human, bovine,rat, mouse, pig, or dog phoenixin propeptide, levels of chemicallysynthesized phoenixin peptides, or the like.

Another broad object of the invention can be to provide purified andisolated chemically synthesized phoenixin peptides which can be utilizedfor the production of polyclonal and monoclonal antibodies which bindone or more of: human, bovine, rat, mouse, pig, dog, or other phoenixinpropeptide, processed forms of human phoenixin propeptide, nativefragments of phoenixin propeptide, or chemically synthesized purifiedand isolated phoenixin peptides.

Another broad object of the invention can be to provide purified andisolated phoenixin peptides which have similar function, similar or newand unexpectedly greater activity or specificity, or both, with respectto the substrates bound as compared to prior known peptides and confersuch function, activity or specificity in a form which can as to certainembodiments omit one or more amino acid residues from known peptides andwhich confer a wide variety of advantages as to ease of production,increased potency, reduced cost, solubility, stability, or the like.

Another broad object of the invention can be to provide kits includingone or more purified and isolated phoenixin peptides and which mayfurther include antibodies raised to one or more phoenixin peptidesuseful in one or more of: radio-immunoassays (“RIA”), enzyme-linkedimmunosorbent assay (“ELISA”), or enzyme immunoassay (“EIA”), or thelike, of tissue or cell homogenates or eluted fractions resulting frompurification and isolation protocols using gel filtration, ion exchangechromatography, reverse phase chromatography, or the like, and for theimmunohistochemical analysis of tissues, or as standards forchromotography or mass spectroscopy, or useful in screening and researchmethods for the determination of specific analogs, agonists,antagonists, partial mimetics, and agents that modulate theirproduction, metabolism, and disposition.

Another broad object of the invention can be a method of regulatingsignal transduction in cells wherein an effective amount or therapeuticamount of one or more purified and isolated phoenixin peptides can becontacted with cells or otherwise administered to modulate or increasethe production of cAMP or Ca²⁺, or both, or to treat disorders relatedto deregulation of signal transduction in cells, tissues or animals.

Naturally, further objects of the invention are disclosed throughoutother areas of the specification, drawings, photographs, and claims.

III. A BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sequence of human cospeptin prepropeptide (Q8N5G0)residues 1-168 (SEQ. ID NO.: 1).

FIG. 2 shows the sequence of phoenixin (1-20) (SEQ. ID NO.: 2) whichcorresponds to the sequence of human cospeptin prepropeptide residues146-165.

FIG. 3 shows the sequence phoenixin (7-21) (SEQ. ID NO.: 3) whichcorresponds to the sequence of human cospeptin prepropeptide residues152-166.

FIG. 4 shows the sequence of phoenixin (7-20) (SEQ. ID NO.: 4) whichcorresponds to the sequence of human cospeptin prepropeptide residues152-165.

FIG. 5 shows the sequence of phoenixin (9-21) (SEQ. ID NO.: 5) whichcorresponds to the sequence of human cospeptin prepropeptide residues154-166.

FIG. 6 shows the sequence of phoenixin (8-20) (SEQ. ID NO.: 6) whichcorresponds to the sequence of human cospeptin prepropeptide residues153-165.

FIG. 7 shows the sequence of phoenixin (1-20) amide (SEQ. ID NO.: 7)which corresponds to the sequence of human cospeptin prepropeptideresidues 146-165 and having a C-terminal amide.

FIG. 8 shows the sequence of phoenixin (8-20) amide (SEQ. ID NO.: 8)which corresponds to the sequence of human cospeptin prepropeptideresidues 153-165 and having a C-terminal amide.

FIG. 9 shows the sequence of phoenixin (7-20) amide (SEQ. ID NO.: 9)which corresponds to the sequence of human cospeptin prepropeptideresidues 152-165 and having a C-terminal amide.

FIG. 10 shows the sequence of pGlu-phoenixin (6-20) amide (SEQ. ID NO.:10) which corresponds to the sequence of human cospeptin prepropeptideresidues 151-165 and having an N-terminal pyroglutamic acid andC-terminal amide.

FIG. 11 shows the sequence of phoenixin (9-20) amide (SEQ. ID NO.: 11)which corresponds to the sequence of human cospeptin prepropeptideresidues 154-165 and having a C-terminal amide.

FIG. 12 shows the sequence of pGlu-phoenixin (9-20) amide (SEQ. ID NO.:12) which corresponds to the sequence of human cospeptin prepropeptideresidues 155-165 and having an N-terminal pyroglutamic acid and having aC-terminal amide.

FIG. 13 shows the sequence of phoenixin (1-20) methylamide (SEQ. ID NO.:13) which corresponds to the sequence of human cospeptin prepropeptideresidues 146-165 and having a C-terminal methylamide.

FIG. 14 shows the sequence of phoenixin (8-20) methylamide (SEQ. ID NO.:14) which corresponds to the sequence of human cospeptin prepropeptideresidues 153-165 and having a C-terminal methylamide.

FIG. 15 shows the sequence of phoenixin (7-20) methylamide (SEQ. ID NO.:15) which corresponds to the sequence of human cospeptin prepropeptideresidues 152-165 and having a C-terminal methylamide.

FIG. 16 shows the sequence of p-glu phoenixin (6-20) methlyamide (SEQ.ID NO.: 16) which corresponds to the sequence of human cospeptinprepropeptide residues 151-165 and having an N-terminal pyroglutamicacid and a C-terminal methylamide.

FIG. 17 shows the sequence of phoenixin (9-20) methylamide (SEQ. ID NO.:17) which corresponds to the sequence of human cospeptin prepropeptideresidues 154-165 and having a C-terminal methylamide.

FIG. 18 shows the sequence of pGlu-phoenixin (9-20) methylamide (SEQ. IDNO.: 18) which corresponds to the sequence of human cospeptinprepropeptide residues 155-165 and having a N-terminal pyroglutamic acidand having a C-terminal methylamide.

FIG. 19 shows the sequence of phoenixin (1-20) ethylamide (SEQ. ID NO.:19) which corresponds to the sequence of human cospeptin prepropeptideresidues 146-165 and having a C-terminal ethylamide.

FIG. 20 shows the sequence of phoenixin (8-20) ethylamide (SEQ. ID NO.:20) which corresponds to the sequence of human cospeptin prepropeptideresidues 153-165 and having a C-terminal ethylamide.

FIG. 21 shows the sequence of phoenixin (7-20) ethylamide (SEQ. ID NO.:21) which corresponds to the sequence of human cospeptin prepropeptideresidues 152-165 and having a C-terminal ethylamide.

FIG. 22 shows the sequence of pGlu-phoenixin (6-20) ethylamide (SEQ. IDNO.: 22) which corresponds to the sequence of human cospeptinprepropeptide residues 151-165 and having a N-terminal pyroglutamic acidand having a C-terminal ethylamide.

FIG. 23 shows the sequence of phoenixin (9-20) ethylamide (SEQ. ID NO.:23) which corresponds to the sequence of human cospeptin prepropeptideresidues 154-165 and having a C-terminal ethylamide.

FIG. 24 shows the sequence of pGlu-phoenixin (9-20) ethylamide (SEQ. IDNO.: 24) which corresponds to the sequence of cospeptin prepropeptideresidues 155-165 and having an N-terminal pyroglutamic acid and having aC-terminal ethylamide.

FIG. 25 shows the sequence of phoenixin (7-20, dValine (8)) amide (SEQ.ID NO.: 25) which corresponds to the sequence of human cospeptinprepropeptide residues 152-165 and having a d-form of valine at residue153 and having a C-terminal amide.

FIG. 26 shows the sequence of phoenixin (7-20, dProline (10)) amide(SEQ. ID NO.: 26) which corresponds to the sequence of human cospeptinprepropeptide residues 152-165 and having a d-form of proline at residue155 and having a C-terminal amide.

FIG. 27 shows the sequence of phoenixin (7-20, dProline (11)) amide(SEQ. ID NO.: 27) which corresponds to the sequence of human cospeptinprepropeptide residues 152-165 and having a d-form of proline at residue156 and having a C-terminal amide.

FIG. 28 shows the sequence of phoenixin (7-20, dAlanine (12) amide (SEQ.ID NO.: 28) which corresponds to the sequence of human cospeptinprepropeptide residues 152-165 and having a d-form of alanine insubstitution of the glycine at residue 157 and having a C-terminalamide.

FIG. 29 shows the sequence of phoenixin (7-20, dLeucine (13)) amide(SEQ. ID NO.: 29) which corresponds to the sequence of human cospeptinprepropeptide residues 152-165 and having a d-form of luecine at residue158 and having a C-terminal amide.

FIG. 30 shows the sequence of phoenixin (7-20, dValine (15)) amide (SEQ.ID NO.: 30) which corresponds to the sequence of human cospeptinprepropeptide residues 152-165 and having a d-form of valine at residue160 and having a C-terminal amide.

FIG. 31 shows the sequence of phoenixin (7-20, dTryptophan (16)) amide(SEQ. ID NO.: 31) which corresponds to the sequence of human cospeptinprepropeptide residues 152-165 and having a d-form of tryptophan atresidue 161 and having a C-terminal amide.

FIG. 32 shows the sequence of phoenixin (7-20, dProline (19)) amide(SEQ. ID NO.: 32) which corresponds to the sequence of human cospeptinprepropeptide residues 152-165 and having a d-form of proline at residue164 and having a C-terminal amide.

FIG. 33 shows the sequence of phoenixin (7-20, dPhenylalanine (20))amide (SEQ. ID NO.: 33) which corresponds to the sequence of humancospeptin prepropeptide residues 152-165 and having a d-form ofphenylalanine at residue 165 and having a C-terminal amide.

FIG. 34 shows the sequence of phoenixin (7-20, Tic(20)) amide (SEQ. IDNO.: 34) which corresponds to the sequence of human cospeptinprepropeptide residues 152-165 and having a1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid at residue 165 insubstitution of the l-form of phenylalanine and having a C-terminalamide.

FIG. 35 shows the sequence of phoenixin (1-20, dAlanine (1)) amide (SEQ.ID NO.: 35) which corresponds to the sequence of human cospeptinprepropeptide residues 146-165 and having a d-form of alanine at residue146 and having a C-terminal amide.

FIG. 36 shows the sequence of phoenixin (1-20, dAlanine (2)) amide (SEQ.ID NO.: 36) which corresponds to the sequence of cospeptin prepropeptideresidues 146-165 and having a d-form of alanine at residue 147 insubstitution for the l-form glycine and having a C-terminal amide.

FIG. 37 shows the sequence of phoenixin (1-20, dValine (4)) amide (SEQ.ID NO.: 37) which corresponds to the sequence of human cospeptinprepropeptide residues 146-165 and having a d-form of valine at residue149 and having a C-terminal amide.

FIG. 38 shows the sequence of phoenixin (1-20, dValine (8)) amide (SEQ.ID NO.: 38) which corresponds to the sequence of human cospeptinprepropeptide residues 146-165 and having a d-form of valine at residue153 and having a C-terminal amide.

FIG. 39 shows the sequence of phoenixin (1-20, dSerine (17)) amide (SEQ.ID NO.: 39) which corresponds to the sequence of human cospeptinprepropeptide residues 146-165 and having a d-form of serine at residue162 and having a C-terminal amide.

FIG. 40 shows the sequence of phoenixin (1-20, dProline (19)) amide(SEQ. ID NO.: 40) which corresponds to the sequence of human cospeptinprepropeptide residues 146-165 and having a d-form of proline at residue164 and having a C-terminal amide.

FIG. 41 shows the sequence of phoenixin (1-20, dPhenylalanine (20))amide (SEQ. ID NO.: 41) which corresponds to the sequence of humancospeptin prepropeptide residues 146-165 and having a d-form ofphenylalanine at residue 165 and having a C-terminal amide.

FIG. 42 shows the sequence of phoenixin acetyl (1-20) amide (SEQ. IDNO.: 42) which corresponds to the sequence of human cospeptinprepropeptide residues 146-165 and having an N-terminal acetyl andhaving a C-terminal amide.

FIG. 43 shows the sequence of phoenixin acetyl (8-20) amide (SEQ. IDNO.: 43) which corresponds to the sequence of human cospeptinprepropeptide residues 153-165 and having an N-terminal acetyl andhaving a C-terminal amide.

FIG. 44 shows the sequence of phoenixin acetyl (7-20) amide (SEQ. IDNO.: 44) which corresponds to the sequence of cospeptin prepropeptideresidues 152-165 and having an N-terminal acetyl and having a C-terminalamide.

FIG. 45 shows the sequence of phoenixin formyl (1-20) amide (SEQ. IDNO.: 45) which corresponds to the sequence of human cospeptinprepropeptide residues 146-165 and having an N-terminal formyl andhaving a C-terminal amide.

FIG. 46 shows the sequence of phoenixin formyl (6-20) amide (SEQ. IDNO.: 46) which corresponds to the sequence of human cospeptinprepropeptide residues 151-165 and having an N-terminal formyl andhaving a C-terminal amide.

FIG. 47 shows the sequence of phoenixin formyl (7-20) amide (SEQ. IDNO.: 47) which corresponds to the sequence of human cospeptinprepropeptide residues 152-165 and having an N-terminal formyl andhaving a C-terminal amide.

FIG. 48 shows the sequence of phoenixin (1-19) napthalene (SEQ. ID NO.:48) which corresponds to the sequence of human cospeptin prepropeptideresidues 146-164 and having a C-terminal naphthelene.

FIG. 49 shows the sequence of phoenixin (7-19) napthalene (SEQ. ID NO.:49) which corresponds to the sequence of human cospeptin prepropeptideresidues 152-164 and having a C-terminal naphthelene.

FIG. 50 shows the sequence of phoenixin (8-19) napthalene (SEQ. ID NO.:50) which corresponds to the sequence of human cospeptin prepropeptideresidues 153-164 and having a C-terminal naphthelene.

FIG. 51 shows the sequence of pGlu-Phoenixin (6-19) napthalene (SEQ. IDNO.: 51) has a sequence located the primary sequence of human cospeptinprepropeptide residues 151-164, pyroglutamic acid at N-terminal andhaving a C-terminal naphthelene.

FIG. 52 shows the sequence of phoenixin (9-19) napthalene (SEQ. ID NO.:52) which corresponds to the sequence of human cospeptin prepropeptideresidues 154-164 and having a C-terminal naphthelene.

FIG. 53 shows the sequence of pGlu-phoenixin (9-19) napthalene (SEQ. IDNO.: 53) which corresponds to the sequence of human cospeptinprepropeptide residues 155-164 and having a pyroglutamic acid at theN-terminal and having a C-terminal naphthelene.

FIG. 54 shows the sequence of Phoenixin (1-20, tryptophan (20)) (SEQ. IDNO.: 54) which corresponds to the sequence of human cospeptinprepropeptide residues 146-165 and having the phenylalanine at residue165 substituted with tryptophan.

FIG. 55 shows the sequence of hoenixin (7-20, tryptophan (20)) (SEQ. IDNO.: 55) which corresponds to the sequence of human cospeptinprepropeptide residues 152-165 and having the phenylalanine at residue165 to be substituted with tryptophan.

FIG. 56 shows the sequence of phoenixin (8-20, tryptophan (20)) (SEQ. IDNO.: 56) which corresponds to the sequence of human cospeptinprepropeptide residues 153-165 and having the phenylalanine at residue165 to be substituted with tryptophan.

FIG. 57 shows the sequence of pGlu-phoenixin (5-20, tryptophan (20))(SEQ. ID NO.: 57) which corresponds to the sequence of human cospeptinprepropeptide residues 151-165, pyroglutamic acid at N-terminal andhaving the phenylalanine at residue 165 to be substituted withtryptophan.

FIG. 58 shows the sequence of phoenixin (9-20, tryptophan (20)) (SEQ. IDNO.: 58) which corresponds to the sequence of human cospeptinprepropeptide residues 154-165 and having the phenylalanine at residue165 to be substituted with tryptophan.

FIG. 59 shows the sequence of pGlu-phoenixin (9-20, tryptophan (20))(SEQ. ID NO.: 59) which corresponds to the sequence of human cospeptinprepropeptide residues 155-165, pyroglutamic acid at N-terminal andhaving the phenylalanine at residue 165 to be substituted withtryptophan.

FIG. 60 shows the sequence of phoenixin (7-20, dTryptophan (11)tryptophan (20)) (SEQ. ID NO.: 60) which corresponds to the sequence ofhuman cospeptin prepropeptide residues 152-165 and having the D-isoformof tryptophan at residue 161 and having the phenylalanine at residue 165to be substituted with tryptophan.

FIG. 61 is a first RP-HPLC separation plot which shows the elution ofimmunoreactive peptides (peaks above the baseline) resulting fromapplication of RP-HPLC to the immunoreactive fractions obtained fromsize fractionation purification of rat tissue homogenates.

FIG. 62 is a mass spectrum profile resulting from mass spectroscopy ofthe fraction including the peak eluting at about 26.5 minutes in thefirst RP-HPLC separation of FIG. 61.

FIG. 63 is a mass spectrum profile resulting from mass spectroscopy inhigh voltage mode of the fraction eluted fraction at about 27 minutes inthe first RP-HPLC separation of FIG. 61.

FIG. 64 is a second RP-HPLC separation plot which shows the elution ofpeptides (peaks above the baseline) resulting from application ofRP-HPLC to the immunoreactive fractions obtained by the above describedfirst RP-HPLC separation of FIG. 61.

FIG. 65 is a mass spectrum profile resulting from mass spectroscopy ofthe fraction including the peak eluting at about 26.5 minutes in thesecond RP-HPLC separation of FIG. 64.

FIGS. 66A through 66D are images of tissue sections of rat medulla onglass slides fixed and immunostained using the anti-phoenixin antibodiesraised against AGIVQEDVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 7) as shown inFIG. 7.

FIGS. 67A through 67D are images of tissue sections of rat forebrain onglass slides fixed and immunostained using the anti-phoenixin antibodiesraised against AGIVQEDVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 7) shown in FIG.7.

FIGS. 68A through 68F are images of tissue sections of rat spinal cordon glass slides fixed and immunostained using the anti-phoenixinantibodies raised against AGIVQEDVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 7)showed in FIG. 7.

FIG. 69 is a bar graph which plots the tissue distribution andconcentration of native phoenixin peptides corresponding toAGIVQEDVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 7) and DVQPPGLKVWSDPF-NH2 (SEQ.ID NO.: 9) as shown in FIG. 7 and FIG. 9.

FIG. 70 is a bar graph compares the production of cAMP in rat pituitarycells challenged with AGIVQEDVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 7) as shownin FIG. 7, DVQPPGLKVWSDPFG (SEQ. ID NO.: 3) as shown in FIG. 3,DVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 9) as shown in FIG. 9, and a phosphatebuffered saline (“PBS”) control.

FIG. 71 shows the alignment of the region of phoenixin (1-20), phoenixin(7-20), and phoenixin (7-21) in different species of animals. From thesequence alignment, the sequence of phoenixin (1-20) in theprepropeptides is identical between the species of human, bovine, rat,and mouse. The peptide of phoenixin (1-20) in the species of canis andpig have one residue difference which is a substitution of valine orisoleucine for the residue of isoleucine or valine.

IV. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As can be easily understood from this description, the basic concepts ofthe present invention may be embodied in a variety of ways. It involvesmethods of producing and using purified and isolated polypeptides andpeptide mimetics which are ligands for protein coupled receptors inregulation of cellular function in gastrointestinal, cardiovascular,hypothalamus-pituitary axis, and central nervous system tissues.

Now referring primarily to FIG. 1, the human phoenixin propeptide(Q8N5G0) of 168 amino acids (SEQ. ID NO.: 1) can be processed to variouspeptide forms including: AGIVQEDVQPPGLKVWSDPF (SEQ. ID NO.: 2) 20 aminoacid residues in length as shown in FIG. 2; DVQPPGLKVWSDPFG (SEQ. IDNO.: 3) 15 amino acid residues in length as shown in FIG. 3;DVQPPGLKVWSDPF (SEQ. ID NO.: 4) 14 amino acid residues in length asshown in FIG. 4, QPPGLKVWSDPFG (SEQ. ID NO.: 5) 13 amino acid residuesin length as shown in FIG. 5, and VQPPGLKVWSDPF (SEQ. ID NO.: 6) 13amino acid residues in length as shown in FIG. 6.

The native phoenixin peptides, as above described, may be in the form ofa free acid at the carboxyl terminus or may be amidated in the form ofAGIVQEDVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 7) as shown in FIG. 7,VQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 8) as shown in FIG. 8, or asDVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 9) as shown in FIG. 9.

Certain residues of the phoenixin peptides (whether free acid or amide)may be L-isoform amino acid or D-isoform amino acid. For example,DVQPPGLKVWSDPFG-NH2 (SEQ. ID NO.: 9) as shown in FIG. 9, andDVQPPGLKVdWSDPF-NH2 (SEQ. ID NO; 31) as shown in FIG. 31.

Phoenixin transcripts can be expressed in human tissues of thegastrointestinal tract, hypothalamus, medulla, forebrain, heart,pituitary, kidney, pancreas, liver, spleen, thymus, and other tissues.

Prediction of Phoenixin Sequences

A peptide library was designed from potential mono- or di-basic cleavagesites in about 100 known preprohormone phoenixin sequences. Candidatesfor peptide synthesis were selected based upon the expectation that thepreprohormone phoenixin protein may be processed in-vivo at a monobasicresidue such as Arg or Lys, dibasic residue pair such as Arg-Arg, amultibasic cleavage site, or the triplet Gly-Arg-Arg which uponproteolytic cleavage can generate the in-vivo processed forms of humanphoenixin, or other forms suitable for antibody production for thecapture of the processed forms of human phoenixin. The modeling resultedin the primary sequence of candidates from which the following wereselected for chemical synthesis: AGIVQEDVQPPGLKVWSDPF (SEQ. ID NO.: 2),DVQPPGLKVWSDPFG (SEQ. ID NO.: 3) and DVQPPGLKVWSDPF (SEQ. ID NO.: 4).

Production of Phoenixin Peptides

Now referring primarily to FIGS. 2-60, once the primary sequence of SEQ.ID NO.: 2, SEQ. ID NO.: 3, and SEQ. ID NO. 4 were determined bymodeling, the corresponding C-terminal free acid (SEQ ID. NOS.: 2through 6), C-terminal amide (SEQ ID NOS.: 7 through 12), C-terminalmethylamide (SEQ. ID NOS. 13 through 18), C-terminal ethylamide (SEQ. IDNOS. 19 through 24), D form amino acid peptides (SEQ. ID. NOS. 24through 41 and 60), N-terminal acetyl (SEQ. ID NOS. 42 through 44),N-terminal formyl (SEQ. ID NOS. 45-47), C-terminal naphthalene (SEQ. IDNOS. 48-50 and 52), and tryptophan substituted for phenylalanine (SEQ.ID. NOS. 54 through 60) peptides were chemically synthesized chemicallyusing fluorenyloxymethylcarbonyl (FMOC) amino acids ortertbutyloxymethylcarbonyl (BOC) amino acids either with an automatedpeptide synthesizer, such as Ranin Instruments Symphony-Multiplexpeptide synthesizer according to the manufacturer's protocol, ormanually as understood by techniques well known to those skilled in theart. See also Solid Phase Peptide Synthesis: A practical approach, E.Atherton and R. C. Sheppard, IRL Press, Oxford, England, herebyincorporated by reference.

Certain phoenixin peptides modified to provide one or more of:N-terminal acetyl, N-terminal formyl, N-terminal pyroglutamic acid,C-terminal amide, C-terminal methyl amide, C-terminal ethylamide,C-terminal naphthalene, C-terminal tryptophan, substitution of one ormore L isoform amino acid residues for the same D isoform amino acid canhave one more of the advantages of being more stable, more soluble, orhave a greater potency as compared to the corresponding original nativepeptide. See for example, Wei E. T et al., Peptides. 1998;19(7):1183-90, hereby incorporated by reference.

The resulting mixture of polypeptides from the chemical synthesis can bepurified and isolated from one another by reverse phase (“RP”) highpressure liquid chromotography (“HPLC”) using columns packed with silicahaving a pore of between 80 angstrom (“Å”) and 300 Å with any one of aC-4, C-8, or C-18 ligand attached. The columns were equilibrated with0.1% trifluoroacetic acid in water at a flow rate dependent on columnsize, as would be understood by those having ordinary skill in the art.The synthetic peptide mixtures were applied to the reverse phase HPLCcolumns and eluted with 0.1% trifluoroacetic acid in acetonitrile usinga gradient of about 0% to about 60% over a period of about 1 hour.Fractions were collected at about 0.5 minute intervals. Fractions weresubsequently analyzed for homogeneity by re-application and elution fromthe reverse phase HPLC system, mass spectrometry, SDS-PAGE, or automatedEdman degradation on a Perkin Elmer/Applied Biosystems Model 470Aprotein sequencer in accordance with the manufacturer's protocol.

The invention further encompasses purified and isolated peptidessubstantially similar to one or more of the phoenixin peptides shown inFIGS. 2-60 (SEQ. ID NOS.: 2 through 60) which retain the function tomodulate intracellular cyclic adenosine monophosphate (“cAMP”)concentration in rat pituitary cells. As non-limiting examples, silentsubstitutions of residues wherein the replacement of the residue withstructurally or chemically similar residue which does not significantlyalter the structure, conformation, or activity of the polypeptide. Suchsilent substitutions are intended to fall within the scope of the claimswhich may be filed in a subsequent non-provisional patent application.As such, the invention and this description is understood to furtherinclude peptides related to SEQ. ID NOS.: 2 through 60 wherein one ormore residues is removed from either end or both ends, or from aninternal region, or wherein one or more residues is added to either endor both ends, or to an internal location in a peptide, or peptideshaving chemical moieties or residues added for chemical orradiolabeling, such as, an added tyrosine for ¹²⁵iodine labeling.Similarly, the N-terminus can be prepared as amino, acetyl, formyl, orleft with a residual FMOC or BOC group intact. The C-terminus can beleft bound to the resin, or cleaved as a carboxyl or an amide by choiceof the corresponding 4-hydroxymethyl-phenylacetamidomethyl (“PAM”) resinor 4-methylbenzhydrylamine hydrochloride salt (“MBHA”) resin. TheC-terminus can be modified to provide a methyl amide, ethyl amide,naphthalene, or other moiety.

Production of Anti-Phoenixin and Phoenixin Peptide Antibodies

Antibodies were raised against each of the chemically synthesizedpurified and isolated peptides corresponding to SEQ. ID.: 3, SEQ. IDNO.: 7, SEQ. ID NO.: 9, SEQ. ID NO.: 10 and SEQ. ID NO.: 11. Antibodieswere prepared in accordance with conventional methods, where thechemically synthesized peptide is used as an immunogen conjugated toknown immunogenic carriers, such keyhole limpet hemocyanin (“KLH”), thesurface antigen of the hepatitis-B-virus (“HBsAg”), other viral oreukaryotic proteins, or the like. Various adjuvants may be employed,with a series of injections, as appropriate. For monoclonal antibodies,after one or more booster injections, the spleen can isolated, thelymphocytes immortalized by cell fusion, and then screened for highaffinity antibody binding. The immortalized cells, (hybridoma),producing the desired antibodies may then be expanded. For a moredetailed description, see Monoclonal Antibodies: A Laboratory Manual,Harlow and Lane eds., Cold Spring Harbor Laboratories, Cold SpringHarbor, N.Y., 1988, hereby incorporated by reference.

Isolation of Native Phoenixin Peptides

Homogenates were prepared from heart tissues of adult rats. Thesupernatant of the rat heart homogenates were passed through C18 columnextraction cartridges and P6 gel filtration to further purify nativepeptide candidates. The immunoreactive fractions as determined byconventional enzyme immunoassay (“EIA”) in which labeled chemicallysynthesized peptides as described above compete with unlabeled nativephoenixin peptide for a limited quantity of the anti-phoenixin peptideantibodies, produced as above described. The label can be biotin complexwhich by reaction with streptavidin horseradish peroxidase and sequentreaction of the horseradish peroxidase with colormetric or fluorescencesubstrates can be quantitated. Immuno-reactive fractions were furtherpurified either by P6 size fractionation gel (Bio-Rad laboratory,Hercules, Calif.) or by ion exchange by application to carboxylmethylcellulose (“CMC”) resin and elution with 0.2 M ammonium acetate. Sincethe P6 size fractionation gave the best immunoreactive results, thisimmunoreactive fractions were then further purified by a first RP-HPLCseparation as above described and the resulting immunoreactive fractionsfurther purified by a subsequent second RP-HPLC separation, as abovedescribed.

Now referring primarily to FIG. 61, a first RP-HPLC separation plotshows the elution of peptides (peaks above the baseline) resulting fromapplication of RP-HPLC to the immunoreactive fractions obtained by theabove described ion exchange procedure. The eluted fractions containingpeptides were assayed by the EIA procedure above described and the levelof immunoreactivity superimposed over the first RP-HPLC plot showingthat the eluted fractions corresponding with the peaks occurring atabout 26.5 minutes and about 27 minutes respectively contain nativephoenixin peptides which bind the corresponding anti-peptide antibodiesproduced as above described.

Now referring primarily to FIG. 62, which shows the mass spectrumresulting from mass spectroscopy of the fraction containing the peptideseluting at about 26.5 minutes in the first RP-HPLC separation. The massspectrum achieved by matrix-assisted laser desorption/ionization time offlight (“MALDI-TOF”) shows that the fraction corresponding to the peakeluted at about 26.5 minutes contains native phoenixin peptidesDVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 9), QPPGLKVWSDPF-NH2 (SEQ. ID NO.: 11)and AGIVQEDVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 7).

Now referring primarily to FIG. 63, which shows the mass spectrumresulting from mass spectroscopy of the fraction containing the peakeluting at about 27 minutes in the first RP-HPLC separation. The massspectrum by high voltage power in MALDI-TOF shows that the fractioncontaining the peak eluting at about 27 minutes contains several nativephoenixin peptides, QPPGLKVWSDPFG (SEQ. ID NO.: 5), VQPPGLKVWSDPF-NH2(SEQ. ID NO.: 8) DVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 9), DVQPPGLKVWSDPFG(SEQ. ID NO.: 3) and AGIVQEDVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 7).

Now referring primarily to FIG. 64, which shows a second RP-HPLCseparation plot evidencing the elution of peptides (peaks above thebaseline) resulting from application of RP-HPLC to the immunoreactivefractions obtained by the above described first RP-HPLC separation. Theeluted fractions containing peptides were assayed by the EIA procedureabove described and the level of immunoreactivity superimposed over thefirst RP-HPLC plot showing that the eluted fractions corresponding withthe peak occurring at about 26 to 26.5 minutes contain native phoenixinpeptides which bind the corresponding anti-peptide antibodies, as abovedescribed.

Now referring primarily to FIG. 65, which shows the mass spectrumresulting from mass spectroscopy of the fraction eluting at about 26 to26.5 minutes in the second RP-HPLC separation. The mass spectrum showsthat the eluted fraction corresponding to the peak at about 26 to 26.5minutes contains native phoenixin peptides DVQPPGLKVWSDPFG (SEQ. ID NO.:3) and DVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 9).

Detection of Phoenixin Peptide in Tissue Sections

Now referring primarily to FIGS. 66A through 66D each of which showtissue sections of rat medulla on glass slides fixed and immunostainedby conventional immunohistochemical staining procedures using theanti-phoenixin antibodies raised against AGIVQEDVQPPGLKVWSDPF-NH2 (SEQ.ID NO.: 7) and DVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 9).Phoenixin-immunofluorescent cell processes are noted in the spinaltrigeminal tract (Sp5) and vagal afferents in FIG. 66A; an enlarged areaof spinal trigeminal tract and vagal afferents in 66B; immunofluorescentcell processes in the medial nucleus of solitary tract (SolM) andcentral nucleus of solitary tract (SolC) in FIG. 66C; immunofluorescentcell processes projecting from the spinal trigeminal tract to thenucleus of ambiguus (nAmb) in FIG. 66D. Scale bar: A, 250 μm; B, C andD, 100 μm.

Generally, immunohistochemical staining on frozen tissue sectionsincludes establishing the tissue sections on glass slides. Fixing thetissue sections with a suitable fixative such as pre-cooled acetone(−20° C.) for 10 min. The fixative can be poured off and the residueacetone evaporated. The slides can be rinsed with a buffer such as 10 mMphosphate buffered saline (PBS) at a neutral pH for 2 changes, 5 mineach. The slides can be incubated in about 0.3% H2O2 solution in PBS atroom temperature for 10 minutes to block endogenous peroxidase activity.The slides are subsequently rinsed in 300 ml PBS for 2 changes, 5 mineach. An optional blocking buffer can be used including for example 10%normal goat serum in PBS onto the tissue sections and incubated at roomtemperature for 1 hour. Apply diluted primary antibody raised againstSEQ. ID NO.: 7 and SEQ. ID NO.: 9 in antibody dilution buffer, of 0.5%bovine serum albumin in PBS to the sections on the slides and incubatefor 1 hour at room temperature or overnight at 4° C. Rinse the slides inabout 300 ml PBS for 2 changes, 5 min each. Apply 100 μl anappropriately diluted biotinylated secondary antibody in the antibodydilution buffer to tissue sections on the slides and incubate at roomtemperature for about 30 min. Rinse the slides in 300 ml PBS for 2changes, 5 min each. Add 100 μl pre-diluted horse radish conjugatesusing the antibody dilution buffer to the sections on the slides andincubate in a humidified chamber at room temperature for 30 minprotected from light. Rinse the slides in about 300 ml PBS for 2changes, 5 min each. Apply about 100 μl 3,3″-diaminobenzidine (“DAB”)substrate solution freshly made just before use: 0.05% DAB-0.015% H2O2in PBS to the sections on the slides to reveal the color of the antibodystaining. Allow the color development for <5 min until the desired colorintensity is reached. Wash slides in 300 ml PBS for 2 changes 5 mineach. Optionally, counter stain slides by immersing sides in hematoxylinfor 1-2 min. Rinse the slides in running tap water for >15 min.Dehydrate the tissue slides through 4 changes of alcohol (95%, 95%, 100%and 100%), 5 min each. Clear the tissue slides in 3 changes of xyleneand coverslip using mounting solution. Observe the color of the antibodystaining in the tissue sections under microscopy.

Now referring to FIGS. 67A through 67D, the immunoactivity of nativePhoenixin peptides in tissue samples of the rat forebrain prepared asabove-described is shown. Phoenixin-immunoreactive cell bodies aredetected in the caudate putamen (CPu) in FIG. 67A; fine cell processescan also be seen in the CPu in FIG. 67B; amygdala in FIG. 67C, andperiventricular nucleus (Pe) in FIG. 67 D. Scale bar: A, 100 μm; B, Cand D, 50 μm.

Now referring to FIGS. 68A through 68F, the immunoactivity of nativephoenixin peptides in tissue sections of rat spinal cord prepared asabove-described is shown. Phoenixin-immunofluorescence occurs in thesuperficial dorsal horn of cervical (FIGS. 68A and 68B), thoracic (FIGS.68C and 68D), and lumbar (FIG. 68E) segments. FIG. 68F shows a lumbarsection processed with phoenixin-antiserum pre-absorbed with the peptide(1 μg/ml overnight; immuno-fluorescence is not detected in the dorsalhorn. Scale bar: A, C, E and F, 250 μm; B and D, 100 μm.

Detection of Phoenixin Peptides in Tissue Homogenate Extracts And BloodPlasma

Now referring primarily to FIG. 69, which shows a bar graph that plotsthe tissue distribution and concentration of native phoenixin peptidescorresponding to AGIVQEDVDVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 7) andDVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 9). Homogenates of porcine or bovine orrat tissue of heart, lung, kidney, spinal cord, small intestine, liver,pancreas, hypothalamus, spleen, and thymus were prepared asabove-described and the resulting fractions were processed by theabove-described peptides extraction procedure and were assayed byradio-immunoassay (“RIA”) or Enzyme Immunoassay (EIA) using antibodiesraised to AGIVQEDVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 7). The antibodiesraised to AGIVQEDVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 7) were also shown tobe 100% cross-reactive with DVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 9) but lessthan 0.5% cross-reactive to DVQPPGLKVWSDPFG (SEQ. ID NO.: 3). Theintra-assay variability was about 5% with a detection limit of about 34picograms/milliliter (“pg/mL”) and an EC50 of about 200 pg/ML. Proteinconcentrations were determined by bicinchoninic acid (“BCA”) proteinassay in accordance to the protocol of the manufacturer ThermoScientific, Rockford, Ill. using bovine serum albumin (“BSA”) as astandard. Data are expressed as the mean±SEM of the results from threeduplicate assays in immunoreactive phoenixin peptide per milligram ofprotein.

As shown in FIG. 69 and Table 1 below, tissues of the liver, pancreas,spleen, kidney and thymus produce substantially less immuno-reactivephoenixin peptides than the tissues of the heart and hypothalamus. Usingthe Fluorescence Phoenixin Enzyme-immunoassay, the phoenixin peptidelevel in human blood plasma without C18 extraction has been determinedto be about 35.5±1.72 pg/ml.

TABLE 1 Tissue Level of Phoenixins Detected by Fluorescent PhoenixinEnzyme-Immunoassay Tissue Homogenates (species) Concentration (pg/mgtissue protein) Cerebrum (rat) Not Dectected Cerebellum (rat)  0.051 ±0.002 Hypothalamus (rat) 363.292 ± 0.384  Hippocampus (rat) Not DetectedPons (rat)  0.218 ± 0.006 Pituitary (rat) 12628.782 ± 505.026  Heart(rat) 1360.539 ± 115.917 Lung (rat) 1924.667 ± 153.15  Stomach (porcine) 520.446 ± 119.702 Small Intestine (rat) 122302.492 ± 18315.776 Kidney(rat)  8530.822 ± 1207.631 Spleen (rat)  5.393 ± 0.067 Pancreas (rat)29.128 ± 0.434 Liver (rat) 585.518 ± 48.389 Ovary (rat) 12.992 ± 0.009Liver (porcine) 0.0887 ± 0.005

Effects of Phoenixin Peptides on Pituitary Cells

Now referring primarily to FIG. 70, a bar graph compares the productionof cAMP in rat pituitary cells challenged with AGIVQEDVQPPGLKVWSDPF-NH2(SEQ. ID NO.: 7) as shown in FIG. 7, DVQPPGLKVWSDPFG (SEQ. ID NO.: 3) asshown in FIG. 3, DVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 9) as shown in FIG. 9and a PBS control.

Rat pituitary adenoma cells, RC-4B/C(CRL-1903; ATCC, Manassas, Va.,USA), were cultured in Dulbecco's Modified Eagle's Medium and MinimumEssential Alpha Medium (Invitrogen, CA, USA) supplemented with 0.01 mMnon-essential amino acids, 15 mM HEPES, 2.5 ng/ml epidermal growthfactor, and dialyzed, heat-inactivated fetal bovine serum (“FBS”) at 37°C. in a humidified cell incubator containing 5% carbon dioxide (“CO₂”).After 2 days of cell cultured in 24 wells plate, cell were equilibratedfor 2 hours in serum-free medium and then incubated with 0.1 mM3-isobutyl-1-methylxanthine (“IBMX”) in serum-free medium 30 min again.Then, cells were challenged with either 100 nM ofAGIVQEDVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 7), DVQPPGLKVWSDPFG (SEQ. ID NO.:3), DVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 9), or a PBS-control in thepresence of IBMX and incubated for 30 min. After incubation, supernatantmedium was then aspirated and the cells in each well were extracted by70% cold ethanol. Alcohol was evaporated in a vacuum concentrator (PN:AES 2000, Savant, Hicksvile, N.Y., USA) Thereafter, cAMP content wasdetermined by using a cAMP Biotrak enzyme immunoassay kit (GEhealthcare-Amersham, Piscataway, N.J., USA) in accordance with theprotocol of the manufacturer.

Also, rat pituitary adenoma cells, RC-4B/C cells, were used in the assayof radioligand binding. For the binding displacement, cells wereincubated for 30 min with 50 pM 1251-Y0-Phoenixin-20 (amide SEQ. ID NO.:7) in the absence or presence of increasing concentrations of un-labeledPhoenixin-20 amide (amide SEQ. ID NO.: 7) or phoenixin-14 amide (amideSEQ. ID NO.: 9). Nonspecific binding was defined as total binding in thepresence of 1 μM unlabeled Phoenixin-20 amide (amide SEQ. ID NO.: 7) orphoenixin-14 amide (amide SEQ. ID NO.: 9). After termination of thebinding reaction by washing the cells with 1 ml of cold PBS, cells weresolubilized with 0.5 ml of 1% SDS, and radioactivity was detected in agamma counter. From non-linear curve fitting, the IC50 for Phoenixin-20amide is 21.5 nM and for Phoenixin-14 amide is 17.9 nM.

As indicated in FIG. 70, AGIVQEDVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 7),DVQPPGLKVWSDPFG (SEQ. ID NO.: 3), DVQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 9)can increase the intracellular cAMP production in rat pituitary adenomacells greater than two fold and even three-fold over the PBS control.Data are expressed as a percentage of the control value (PBS-control,100%; 4.94±0.5 pmol/mg protein).

Uses of Phoenixin Peptides

Phoenixin peptides SEQ ID NOS.: 2 through 60 can be utilized in fourgeneral areas. Firstly, as antigens in the form of one or more Phoenixinpeptides SEQ ID NOS.: 2 through 60 which can be utilized in the assaysabove or below described or to raise monoclonal or polyclonalantibodies, as above described, The resulting monoclonal or polyclonalantibodies can be useful in binding one or more of the phoenixinpropeptide, phoenixin peptides, or the like.

Secondly, as molecular tools or reagents in kits including one or morechemically synthesized phoenixin peptides SEQ ID NOS.: 2 through 60which can be accompanied by antibodies raised to one or more of thephoenixin peptides. The kits can be useful for example inradio-immunoassays (“RIA”), enzyme-linked immunosorbent assay (“ELISA”),or enzyme immunoassay (“EIA”), or the like, of tissue or cellhomogenates or eluted fractions resulting from purification protocolsusing gel filtration, ion exchange chromatography, reverse phasechromatography, immunoprecipitation or the like, and for theimmunohistochemical analysis of tissues, or as standards forchromatography or mass spectroscopy, or as a biomarker in the diseasescreening.

Thirdly, as a diagnostic tool in clinical usage for assessment ofcertain diseases. Since native phoenixin peptides can be functionalpeptides in human or animal physiology, the absence or abnormal levels(whether abnormally high or low compared to normal values) of phoenixinpeptides can correlate with other physiological factors or symptomswhich indicate specific diseases. The presence of a certain amount ofPhoenixin peptide in the blood or tissues can be used as an indicationor as a guide index for certain medical treatments.

Fourthly, the phoenixin peptides can play a functional role in normalphysiology. Over-expression or under-expression of native phoenixinpeptides can result in the related disease or dysfunction. Therefore,administration of one or more phoenixin peptide(s) or phoenixin peptidesto the animal or human body, or contact with cells that bind,competitively bind, transfer, or otherwise utilize phoenixin peptides toregulate or modulate a physiological pathway can reverse or correct thedisease state. In order to maximize therapeutic effectiveness,administration of one or more phoenixin peptides may be accomplishedthrough different methods such as intravenous, intramuscular,sub-cutaneous, or the like, alone or in conjunction with otherpharmaceutical reagents in amounts sufficient to generate a therapeuticeffect, such as cardiovasular response to lower blood pressure, emptythe bowel, release gonadotropins, or the like. As but one example, oneor more of phoenixin SEQ. ID NOS.: 2 to 60 can be administered toanimals to achieve a decrease in blood pressure. Additionally, one ormore Phoenixin peptides SEQ. ID NOS.: 2 to 60 can be administered toanimals to regulate cell signal and in particular phoenixin peptidesAGIVQEDVQPPGLKVWSDPF (SEQ. ID NO.: 2), DVQPPGLKVWSDPFG (SEQ. ID NO.: 3),DVQPPGLKVWSDPF (SEQ. ID NO.: 4), QPPGLKVWSDPFG (SEQ. ID NO.: 5)VQPPGLKVWSDPF (SEQ. ID NO.: 6), AGIVQEDVQPPGLKVWSDPF-NH2 (SEQ. ID NO.:7), VQPPGLKVWSDPF-NH2 (SEQ. ID NO.: 8), DVQPPGLKVWSDPF-NH2 (SEQ. ID NO.:9), DVQPPGLKVdWSDPF-NH2 (SEQ. ID NO.: 31), DVQPPGLKVWSDdPF-NH2 (SEQ. IDNO.: 32) and DVQPPGLKVdWSDPW-NH2 (SEQ. ID NO.: 60) can be utilized tomodulate cAMP production in cells.

Now referring to FIG. 71, the alignment of the region of phoenixin(1-20), phoenixin (7-20), and phoenixin (7-21) is shown in differentspecies of animals. The sequence alignment evidences that the sequenceof phoenixin (1-20) in the prepro-proteins is identical between thespecies of human, bovine, rat, and mouse. phoenixin (1-20) in thespecies of canis and pig have one residue difference which is onesubstitution of valine or isoleucine for the residue of isoleucine orvaline. Accordingly, the sequences, synthesis or isolation of phoenixinpeptides, analysis, and function as above described can be conservedbetween species.

Now referring to the Figures in general and the description of theFigures above, any reference to human phoenixin or human phoenixinpeptides along with the residue position identifiers in the propeptidehuman phoenixin (Swiss-Prot: Q8N5G0) are for alignment reference onlyand it is not intended that these references admit or suggest that anyof the phoenixin peptides shown in the Figures or described above wereidentified in the prior art, occur in nature, or structure or functionof propeptide human phoenixin is similar to the described phoenixinpeptides.

As can be easily understood from the foregoing, the basic concepts ofthe present invention may be embodied in a variety of ways. Theinvention including the best mode involves numerous and variedembodiments of phoenixin/phoenixin peptides useful for the production ofantibodies, diagnostic screening and assays, modulation of cellularcAMP, and treatment of disorders benefited by peptides which canmodulate cAMP, hypertension, and smooth muscle response.

As such, the particular embodiments or elements of the inventiondisclosed by the description including the best mode or shown in thefigures or tables accompanying this application are not intended to belimiting, but rather exemplary of the numerous and varied embodimentsgenerically encompassed by the invention or equivalents encompassed withrespect to any particular element thereof. In addition, the specificdescription of a single embodiment or element of the invention may notexplicitly describe all embodiments or elements possible; manyalternatives are implicitly disclosed by the description and figures.

It should be understood that each element of an apparatus or each stepof a method may be described by an apparatus term or method term. Suchterms can be substituted where desired to make explicit the implicitlybroad coverage to which this invention is entitled. As but one example,it should be understood that all steps of a method may be disclosed asan action, a means for taking that action, or as an element which causesthat action. Similarly, each element of an apparatus may be disclosed asthe physical element or the action which that physical elementfacilitates. As but one example, the disclosure of “a chemicallysynthesized peptide” should be understood to encompass disclosure of theact of “chemically synthesizing a peptide”—whether explicitly discussedor not—and, conversely, were there effectively disclosure of the act of“chemically synthesizing a peptide”, such a disclosure should beunderstood to encompass disclosure of “a chemically synthesized peptide”and even a “means for chemically synthesizing a peptide.” Suchalternative terms for each element or step are to be understood to beexplicitly included in the description.

In addition, as to each term used it should be understood that unlessits utilization in this application is inconsistent with suchinterpretation, common dictionary definitions should be understood to beincluded in the description for each term as contained in the RandomHouse Webster's Unabridged Dictionary, second edition, each definitionhereby incorporated by reference.

Moreover, for the purposes of the present invention, the term “a” or“an” entity refers to one or more of that entity; for example, “a lightsource” refers to one or more of those light sources. As such, the terms“a” or “an”, “one or more” and “at least one” can be usedinterchangeably herein.

All numeric values herein are assumed to be modified by the term“about”, whether or not explicitly indicated. For the purposes of thepresent invention, ranges may be expressed as from “about” oneparticular value to “about” another particular value. When such a rangeis expressed, another embodiment includes from the one particular valueto the other particular value. The recitation of numerical ranges byendpoints includes all the numeric values subsumed within that range. Anumerical range of one to five includes for example the numeric values1, 1.5, 2, 2.75, 3, 3.80, 4, 5, and so forth. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. When a value is expressed as an approximation by use of theantecedent “about,” it will be understood that the particular valueforms another embodiment.

Thus, the applicant(s) should be understood to claim at least: i) eachof the phoenixin peptides herein disclosed and described, ii) therelated methods disclosed and described, iii) similar, equivalent, andeven implicit variations of each of these devices and methods, iv) thosealternative embodiments which accomplish each of the functions shown,disclosed, or described, v) those alternative designs and methods whichaccomplish each of the functions shown as are implicit to accomplishthat which is disclosed and described, vi) each feature, component, andstep shown as separate and independent inventions, vii) the applicationsenhanced by the various systems or components disclosed, viii) theresulting products produced by such systems or components, ix) methodsand apparatuses substantially as described hereinbefore and withreference to any of the accompanying examples, x) the variouscombinations and permutations of each of the previous elementsdisclosed.

The background section of this patent application provides a statementof the field of endeavor to which the invention pertains. This sectionmay also incorporate or contain paraphrasing of certain United Statespatents, patent applications, publications, or subject matter of theclaimed invention useful in relating information, problems, or concernsabout the state of technology to which the invention is drawn toward. Itis not intended that any United States patent, patent application,publication, statement or other information cited or incorporated hereinbe interpreted, construed or deemed to be admitted as prior art withrespect to the invention.

The claims set forth in this specification, if any, are herebyincorporated by reference as part of this description of the invention,and the applicant expressly reserves the right to use all of or aportion of such incorporated content of such claims as additionaldescription to support any of or all of the claims or any element orcomponent thereof, and the applicant further expressly reserves theright to move any portion of or all of the incorporated content of suchclaims or any element or component thereof from the description into theclaims or vice-versa as necessary to define the matter for whichprotection is sought by this application or by any subsequentapplication or continuation, division, or continuation-in-partapplication thereof, or to obtain any benefit of, reduction in feespursuant to, or to comply with the patent laws, rules, or regulations ofany country or treaty, and such content incorporated by reference shallsurvive during the entire pendency of this application including anysubsequent continuation, division, or continuation-in-part applicationthereof or any reissue or extension thereon.

The claims set forth in this specification, if any, are further intendedto describe the metes and bounds of a limited number of the preferredembodiments of the invention and are not to be construed as the broadestembodiment of the invention or a complete listing of embodiments of theinvention that may be claimed. The applicant does not waive any right todevelop further claims based upon the description set forth above as apart of any continuation, division, or continuation-in-part, or similarapplication.

1. A purified and isolated polypeptide, comprising of any one of: SEQ.ID NO: 2, SEQ. ID NO: 3, SEQ. ID NO: 4, SEQ. ID NO: 5, SEQ. ID NO: 6,SEQ. ID NO: 7, SEQ. ID NO: 8, SEQ. ID NO: 9, SEQ. ID NO: 10, SEQ. ID NO:11, SEQ. ID NO: 12, SEQ. ID NO: 13, SEQ. ID NO: 14, SEQ. ID NO: 15, SEQ.ID NO: 16, SEQ. ID NO: 17, SEQ. ID NO: 18, SEQ. ID NO: 19, SEQ. ID NO:20, SEQ. ID NO: 21, SEQ. ID NO: 22, SEQ. ID NO: 23, SEQ. ID NO: 24, SEQ.ID NO: 25, SEQ. ID NO: 26, SEQ. ID NO: 27, SEQ. ID NO: 28, SEQ. ID NO:29, SEQ. ID NO: 30, SEQ. ID NO: 31, SEQ. ID NO: 32, SEQ. ID NO: 33, SEQ.ID NO: 34, SEQ. ID NO: 35, SEQ. ID NO: 36, SEQ. ID NO: 37, SEQ. ID NO:38, SEQ. ID NO: 29, SEQ. ID NO: 40, SEQ. ID NO: 41, SEQ. ID NO: 42, SEQ.ID NO: 43, SEQ. ID NO: 44, SEQ. ID NO: 45, SEQ. ID NO: 46, SEQ. ID NO:47, SEQ. ID NO: 48, SEQ. ID NO: 49, SEQ. ID NO: 50, SEQ. ID NO: 51, SEQ.ID NO: 52, SEQ. ID NO: 53, SEQ. ID NO: 54, SEQ. ID NO: 55, SEQ. ID NO:56, SEQ. ID NO: 57, SEQ. ID NO: 58, SEQ. ID NO: 59, and SEQ. ID NO: 60.2. The purified and isolated polypeptide of claim 1, consisting of anyone of: the polypeptide consisting of SEQ. ID NO: 2, the polypeptideconsisting of SEQ. ID NO: 3, the polypeptide consisting of SEQ. ID NO:4, the polypeptide consisting of SEQ. ID NO: 5, the polypeptideconsisting of SEQ. ID NO: 6, the polypeptide consisting of SEQ. ID NO:7, the polypeptide consisting of SEQ. ID NO: 8, the polypeptideconsisting of SEQ. ID NO: 9, the polypeptide consisting of SEQ. ID NO:10, the polypeptide consisting of SEQ. ID NO: 11, the polypeptideconsisting of SEQ. ID NO: 12, the polypeptide consisting of SEQ. ID NO:13, the polypeptide consisting of SEQ. ID NO: 14, the polypeptideconsisting of SEQ. ID NO: 15, the polypeptide consisting of SEQ. ID NO:16, the polypeptide consisting of SEQ. ID NO: 17, the polypeptideconsisting of SEQ. ID NO: 18, the polypeptide consisting of SEQ. ID NO:19, the polypeptide consisting of SEQ. ID NO: 20, the polypeptideconsisting of SEQ. ID NO: 21, the polypeptide consisting of SEQ. ID NO:22, the polypeptide consisting of SEQ. ID NO: 23, the polypeptideconsisting of SEQ. ID NO: 24, the polypeptide consisting of SEQ. ID NO:25, the polypeptide consisting of SEQ. ID NO: 26, the polypeptideconsisting of SEQ. ID NO: 27, the polypeptide consisting of SEQ. ID NO:28, the polypeptide consisting of SEQ. ID NO: 29, the polypeptideconsisting of SEQ. ID NO: 30, the polypeptide consisting of SEQ. ID NO:31, the polypeptide consisting of SEQ. ID NO: 32, the polypeptideconsisting of SEQ. ID NO: 33, the polypeptide consisting of SEQ. ID NO:34, the polypeptide consisting of SEQ. ID NO: 35, the polypeptideconsisting of SEQ. ID NO: 36, the polypeptide consisting of SEQ. ID NO:37, the polypeptide consisting of SEQ. ID NO: 38, the polypeptideconsisting of SEQ. ID NO: 39, the polypeptide consisting of SEQ. ID NO:40, the polypeptide consisting of SEQ. ID NO: 41, the polypeptideconsisting of SEQ. ID NO: 42, the polypeptide consisting of SEQ. ID NO:43, the polypeptide consisting of SEQ. ID NO: 44, the polypeptideconsisting of SEQ. ID NO: 45, the polypeptide consisting of SEQ. ID NO:46, the polypeptide consisting of SEQ. ID NO: 47, the polypeptideconsisting of SEQ. ID NO: 48, the polypeptide consisting of SEQ. ID NO:49, the polypeptide consisting of SEQ. ID NO: 50, the polypeptideconsisting of SEQ. ID NO: 51, the polypeptide consisting of SEQ. ID NO:52, the polypeptide consisting of SEQ. ID NO: 53, the polypeptideconsisting of SEQ. ID NO: 54, the polypeptide consisting of SEQ. ID NO:55, the polypeptide consisting of SEQ. ID NO: 56, the polypeptideconsisting of SEQ. ID NO: 57, the polypeptide consisting of SEQ. ID NO:58, the polypeptide consisting of SEQ. ID NO: 59, and the polypeptideconsisting of SEQ. ID NO:
 60. 3. The purified polypeptide of claim 1,wherein said purified and isolated polypeptide has at least oneconservative amino acid substitution.
 4. The purified polypeptide ofclaim 3, wherein said at least one conservative amino acid substitutioncomprises substitution of an L form amino acid residue with a D formamino acid residue.
 5. The purified polypeptide of claim 3, wherein saidpurified and isolated polypeptide has an amide C-terminus.
 6. Thepurified polypeptide of claim 3, wherein said purified and isolatedpolypeptide has an acetyl N-terminus.
 7. The purified polypeptide ofclaim 5, wherein said purified and isolated polypeptide has an amideC-terminus. 8-11. (canceled)